A hierarchical cascade activated by non-canonical Notch signaling and the mTOR Rictor complex regulates neglect- induced death in mammalian cells
Supplementary Materials for Perumalsamy et al
Figure S1 NIC inhibits neglect induced death
(A) HeLa cells were cultured overnight and subsequently the culture medium was
replaced by buffered saline. The cells were harvested at indicated time points and stained
with Hoechst 33342 or with Annexin-V-FITC. Apoptotic nuclear morphology was scored
in cells stained using Hoechst 33342 (filled circles). Annexin V positive cells were
analyzed using flow cytometry (triangles). Data represent the mean + SD from three
separate experiments.
(B) The antibody to Val-1744 specifically recognizes the gamma secretase (S3) cleaved
form of human Notch1. Western blot analysis of Val-1744 levels in HeLa cell lysates, 16
hours after transfection with GFP, Notch Full length (NFL) with or without sJag1. αtubulin was used as the loading control.
(C) HeLa cells transfected with RFP or NIC-RFP were cultured overnight to allow for
expression of the recombinant proteins. Subsequently, spent medium was replaced with
fresh complete medium (nutrient replete) or buffered saline (neglect). After 6 hours,
apoptotic damage was analyzed using the TUNEL assay (green). Cells were also
counterstained with Hoechst 33342 (blue). Representative confocal images are shown.
NIC-RFP is predominantly localized to the nucleus and excluded from heterochromatin.
Scale Bar: 5µm
(D) HeLa cells transfected with GFP or NIC-GFP were cultured overnight. Subsequently
the cells were either cultured in CM (control) or buffered saline (neglect) and apoptotic
damage assessed after 22 hours. The empty vector pCDNA-3.0 was used to equalize the
DNA amount transfected across different groups in all experiments. Data are normalized
to the nutrient replete condition and represent mean + SD from three separate
experiments.
Figure S2 NIC activates AktS473
(A) Western blot analysis to determine the phosphorylation of AktS473 and AktT308 in
HEK cells transfected with Akt-myc + NIC with or without Numb.
(B) Phosphorylation of AktS473 in HEK cells transfected with Akt-myc with or without
Numb, in the absence of NIC.
Figure S3 NIC blocks neglect induced death in a human fibroblast cell line LTG
(A) siRNA-mediated depletion of mTOR (top panel), Raptor (middle panel) and Rictor
(bottom panel) in HEK cells as indicated by western blot analysis for the respective
proteins.
(B) LTG cells were transfected with GFP or NIC-GFP and cultured overnight.
Subsequently, culture medium was replaced with CM (nutrient replete) or buffered saline
(neglect). Apoptotic nuclear damage was assessed in GFP positive cells using Hoechst
33342 six hours later. The data are corrected to the nutrient replete condition and are the
mean + SD from three independent experiments.